A balance between vasoconstrictor and dilator systems plays a critical role in the kidney, setting the blood flow, glomerular filtration rate and sodium excretion. Our goal is to gain a better understanding of hormonal, paracrine and autocrine control of vasomotor tone in the renal microcirculation in health and disease, with particular emphasis on regulation of vascular reactivity and receptor signaling pathways in preglomerular resistance arterioles in genetic hypertension. Our previous studies on hypertensive adult spontaneously hypertensive rats (SHR) indicate that excessive renal vasoconstriction is mediated by an imbalance of actions of the vasoconstrictors Ang II and thromboxane (TxA2) and vasodilator systems (prostanoids, nitric oxide (NO)). Our recent work indicates that Ca2+ and contractile responses are mediated by signaling cascade involving ADP ribosyl cyclase and Ca2+ release from ryanodine receptors (RyR). In new studies, we shall focus on pro-hypertensive events before the development of hypertension which are most likely to be causative. We shall characterize interactions among Ang II, endothelin-1 (ET1), and TxA2 and their stimulation of NAD(P)H oxidase and superoxide anion (O2-) on vasomotor tone and Ca2+ signaling in afferent arterioles of prehypertensive 4-5-wk-old SHR. We hypothesize that exaggerated reductions in renal blood flow (RBF) in SHR are mediated by direct actions of constrictor agents on VSMC, either alone, due to enhanced receptor density or post-receptor signaling, or in concert with deficient buffering by the vasodilator NO. We propose that vasoconstriction favored by O2- is due to interactions with Ca2+ signaling in VSMC plus scavenging of NO. Specific aims will test the hypotheses that: 1) Renal vascular reactivity to Ang II, ET1 and TxA2 is exaggerated in prehypertensive SHR and that the O2-, ADP ribosyl cyclase, RyR and Ca2+ mobilization pathway plays a critical role;2) Ca2+ signaling in afferent arterioles is enhanced in response to Ang II, ET1 and TxA2 and that the O2- / ADP ribosyl cyclase / RyR pathway is central;and 3) mRNA and protein levels of receptors for Ang II, ET1, and TxA2 and NAD(P)H subunits are up-regulated in the preglomerular vasculature of 4-5-wk-old SHR. Innovative complementary in vivo RBF studies of integrative pathophysiology and in vitro studies of cellular effector signal transduction in isolated arterioles will provide insight into pro-hypertensive mechanisms responsible for renal vasoconstriction in prehypertensive SHR.